|
| Status |
Public on Jul 10, 2015 |
| Title |
BMDM_MCMV(pSM3)_Infection_T90 |
| Sample type |
RNA |
| |
|
| Source name |
Bone marrow-derived macrophages, mCMV infected, 90 min
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
gender: male
age: 10-12 weeks
developmental stage: adult
tissue: bone marrow
cell type: bone marrow-derived macrophages (BMDMs)
treatment: murine cytomegalovirus (mCMV) infection
time point: 90 min
|
| Treatment protocol |
Day 7 - media taken off the two wells and new media added (infection) - DMEM/F-12 + 2% FCS+ 10% L929 + pen/strep, mCMV(pSM3) MOI of 1 - 0.5ml/well - for 1 hour (with shaking every 10 minutes). This infection media is taken off and the cells are washed three times in PBS. New media is added, mock interferon-gamma treatment - DMEM/F-12 + 10% FCS + 10% L929 + pen/strep. The media is taken off at the sample timepoint and the cells are lysed in Trizol and stored at -80C.
|
| Growth protocol |
8x10^5 cells were plated out into 2 wells of a 6-well plate. Media - DMEM/F-12 + 10% FCS + 10% L929 + pen/strep. Media re-feed on day 3 and on day 5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PAXgene RNA Extraction: extraction of total RNA from whole blood taken into PAXgene RNA tubes according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Agilent Low RNA Input: Agilent Low RNA Input HALF Fluorescent Linear Amplification. Synthesis of labelled cRNA from 50-500ng total RNA.
|
| |
|
| Hybridization protocol |
Dual hybridisation (Cy3 and Cy5) to Agilent oligo microarrays.
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner according to the Agilent G2565AA/G2565BA Microarray Scanner System Manual.
Images were quantified using Agilent Feature Extraction Software (version 7.5).
|
| Description |
MCMV_T90
|
| Data processing |
Basic processing of the microarray data set includes background correction, log2 transformation, subset normalisation and filtering, using the R limma package. Data quality and consistency was assessed at each stage. After background subtraction in each measurement channel, features resulting in negative expression levels were set to 1. All measurements were transformed to log base 2. Systematic variation between samples was corrected using a subset median normalisation step. For this, we identified 42 features (including duplications) that are positive control or housekeeping genes for the type of biological samples used. The median of these log2 scale features was calculated for 75 test and reference samples, respectively. For all samples, the normalisation constant to be added to each feature on an array was calculated as the difference between that individual array?s positive control probe median and a reference value which was calculated as the arithmetic mean of all 75 positive control probe medians. In order to minimise the number of genes with random expression spikes or invariant expression, we devised a method centered around detection thresholds through ROC analysis. A ROC analysis of a gold-standard set of 42 positive and 111 negative control probes is done separately for each of the 75 samples. A cut-off value for achieving sensitivity >= 80% is determined for each sample. The arithmetic mean of all 75 cut-off values then constitutes the desired detection threshold and is used to construct a binary filter matrix indicating if a gene is above or below that threshold. A gene is excluded from the data set if it does not exceed the detection threshold in 5 consecutive time points in any of the three biological conditions. Of the original 22393 features, 14819 remain.
|
| |
|
| Submission date |
Mar 24, 2013 |
| Last update date |
Jul 10, 2015 |
| Contact name |
Nigel Binns |
| E-mail(s) |
n.binns@ed.ac.uk
|
| URL |
http://www.ed.ac.uk/pathway-medicine
|
| Organization name |
The University of Edinburgh
|
| Department |
Division of Pathway Medicine
|
| Street address |
The Chancellor's Building, 49 Little France Crescent
|
| City |
Edinburgh |
| ZIP/Postal code |
EH16 4SB |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL891 |
| Series (2) |
| GSE42503 |
Timecourse of mCMV Infection of Bone Marrow-Derived Macrophages (BMDMs) |
| GSE42505 |
Interferon-Gamma- and Murine-CMV-Activated Primary Macrophages |
|
